Abstract/Summary Actin cytoskeleton rearrangement, an important determinant of both cellular motility and the maintenance of cellular structure-function, is regulated by members of the Rho family of small GTPases. Rho family GTPase? regulated proteins that catalyze actin fiber polymerization include those of the Wiskott Aldrich family (WASp/ WAVE) proteins acting on their downstream Arp2/3-complex, and formins including the Diaphanous-related formin (DRF) subclass. How DRF activity is controlled is poorly understood compared with WASp/WAVE proteins, however, particularly in relation to cellular functions driven by DRFs that are activated by Rho GTPases. Our primary objective therefore, is to develop new fluorescent biosensor imaging tools for studying the DRFs mammalian Diaphanous formin (mDia)1 and 2, for the first time. Our second objective is to extend these new mDia biosensors and our Rho family GTPase biosensors to multiplex-imaging compatible format through the use of our new near-infrared (NIR) fluorescent proteins (FPs) ideally suited for Frster resonance energy transfer (FRET) applications. The new NIR FRET biosensors developed herein, will for the first time enable simultaneous multiplex imaging and/or perturbation of upstream Rho GTPases and monitoring of mDia1 and 2 activities in the same cell; the new NIR FRET modality will be optimized for simultaneous imaging using traditional cyan fluorescent protein (CFP)?yellow fluorescent protein (YFP) FRET or combination with optogenetic tools that require blue-green light. In summary, our proposed new biosensor technology for mDia1 and 2 will enable for the first time direct, simultaneous monitoring and/or perturbation of Rho GTPase and mDia activities, allowing determination of the specificity of Rho GTPase?initiated signalling dynamics regarding the functions of mDia1 and 2 in living cells. The approaches pursued in this proposal should significantly advance the current state of the art in technology for studying RhoGTPase?mDia biology.